Emergence of antibiotic resistant pathogens is a global healthcare crisis that is forcing physicians to treat common infectious-diseases with ever more potent antibiotics. New strategies are urgently needed for rapid identification of drug resistant healthcare-associated infections and to provide clinicians with real-time information to guide antibiotic selection. The goal of the research described in this collaborative proposal is to develop all of the essential technological components needed to produce a biosensor for rapid high throughput bacterial pathogen identification and antibiotic susceptibility determination. We have assembled a multidisciplinary team to achieve this goal including academic arid industry leaders in the fields of electrochemical sensors (J. W., V.G.) molecular microbiology (D. H.), antimicrobial susceptibility testing (D.B, J.H.), urology (B.C.) and biomathematics (E. L.). The biosensor group has successfully developed an electrochemical sensor for rapid genotypical identification of uropathogens. The electrochemical sensor assay involves "sandwich" hybridization of target 16S rRNA to species-specific capture'and detector probes. The assay can be performed at room temperature and has the sensitivity to defect as few as 200 bacteria. Proof Of concept has already been demonstrated in a clinical study of urine specimens from patients with urinary tract infections. The Research Plan has three Specific Aims. Specific Aim 1 describes methods to enhance the performance of the electrochemical sensor assay through improved control of the surface chemistry and signal amplification techniques. These efforts will result in PCR-like sensitivity without an increase in background signal. Specific Aim 2 describes development of a rapid antimicrobial susceptibility assay. Species specific probes have been developed for most of the pathogens called for in the RFA, these probes will be validated and tested in a rapid test measuring the phenotypic response of clinical isolates to relevant antibiotics. Specific Aim 3 will be to adapt the electrochemical sensor assay to an automated, robotic, high throughput system, which we refer to as the PATHOSENSE instrument. Analytic performance of the PATHOSENSE instrument will be evaluated in a clinical study of patients at high risk for hospital acquired urinary tract infection. As described in the Product Development Plan, these studies will position our industrial partner, GeneFluidics, for near-term deployment of the PATHOSENSE instrument in multiple clinical settings.